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Biomedical subjects

P B Garland

Publications and source records attributed to P B Garland.

At least 19 recordsLinked to original sources
PubMed1991

Chemiosmotic systems in medicine.

The concept of chemiosmotic systems arises from the pioneering work of Peter Mitchell on two fronts. One is concerned with the mechanisms by which molecules are transported across membranes which are generally barriers to such transport. These mechanisms are inevitably molecular, and are now yielding their secrets to a combination of structural protein chemistry and molecular biology. The other front is more physiological, and explores the functional relationships between metabolism and transport. Nevertheless, the two fronts form a continuum of mutually related structure and function. Chemiosmotic systems provide a hierarchy of complexity, starting from say a uniporter reconstituted in a chemically defined bilayer, and proceeding to greater complexity in mitochondria, chloroplasts, eukaryotic and prokaryotic cell membranes, and multicellular systems. Their relationship to medicine is profound, because they provide many opportunities for therapeutic intervention. In this paper I present an overview of chemiosmotic systems at different levels of complexity, both molecular and biological, of their involvements in pathology, and of possible pharmacological treatment or prevention of disease.

Biological Transport, Active↗
PubMed1989

Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells.

Post-embedding immunogold electron microscopy shows several binding sites for cholera toxin in mouse intestinal epithelial cells, particularly in the heterochromatin of the nucleus as well as in the plasma membrane. Anti-ganglioside GM1 antibodies also bound to the nucleus, but did not interfere with the binding of toxin. 125I-labelled toxin bound specifically to a nuclear preparation from rabbit intestinal cells.

Animals↗
PubMed1989

Effect of some polypeptide hormones on the lateral mobility in plasma membranes of rat testes, L-cells and liver. Influence of development and ageing.

1. The effect of different doses of six polypeptide hormones on pyrene diffusion in rat testes and liver plasma membranes was tested. Pyrene mobility was reduced in membranes possessing respective receptors. 2. An incubation time of 15 min of testes plasma membranes with 10(-5) M lutropin (LH) reduced pyrene and diphenyl hexatriene mobilities by 10-20%. 3. The addition of 10(-5) M LH to a suspension of intact L-cells from rats at different ages decreased the diffusion of membrane fluorescently labelled lipids and proteins by ca 60%. Diffusion was measured by fluorescence recovery after photobleaching. 4. Observed LH effects were independent of development and ageing.

Aging↗
PubMed1989

Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments.

Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions. The implications of this finding for cell division and other periplasmic processes are discussed.

Biological Transport↗
PubMed1988

Changes in the organization of the surface membrane upon transformation of cercariae to schistosomula of the helminth parasite Schistosoma mansoni.

Merocyanin 540 (Mc540) is a fluorescent compound which is thought to bind to membranes in which there are substantial amounts of lipid in the lipid-crystalline phase. It is shown here to be of value in detecting the transformation by both mechanical and skin-penetration methods of the cercaria to the schistosomulum. The cercaria does not appear to bind Mc540, but the schistosomulum, binds Mc540 initially, in its anterior region, and at later times over the entire surface. The suggestion that transformation involves changes in the surface membrane lipid phase from gel to liquid-crystalline phase is supported by fluorescence recovery after photobleaching results with 5-N-(octadecanoyl)-amino fluorescein, a lipophilic dye which appears to be immobile in the cercaria, but fully mobile in the 40 min schistosomulum.

Animals↗
PubMed1987

Biophysical properties of the surface lipid of parasitic nematodes.

The biophysical properties of the surface lipid layer (the epicuticle) of living parasitic nematodes (Trichinella spiralis and Toxocara canis) were examined using fluorescent lipid analogues. A variety of such probes were screened, and only 5-N-(octadecanoyl)-aminofluorescein was found to insert into the outer lipid layer. Fluorescence quenching experiments showed that this probe was confined to the surface, and the rate of its lateral diffusion was then measured by Fluorescence Recovery After Photobleaching. This showed that the probe was not free to diffuse within the plane of the epicuticle. This structure is, therefore, extraordinary in its selectivity to lipid probes, and in the restricted lateral mobility of inserted lipid components.

Animals↗
PubMed1986

The lateral diffusion of lipid probes in the surface membrane of Schistosoma mansoni.

The technique of fluorescence recovery after photobleaching was used to measure the lateral diffusion of fluorescent lipid analogues in the surface membrane of Schistosoma mansoni. Our data reveal that although some lipids could diffuse freely others exhibited restricted lateral diffusion. Quenching of lipid fluorescence by a non-permeant quencher, trypan blue, showed that there was an asymmetric distribution of lipids across the double bilayer of mature parasites. Those lipids that diffused freely were found to reside mainly in the external monolayer of the outer membrane whereas lipids with restricted lateral diffusion were located mainly in one or more of the monolayers beneath the external monolayer. Formation of surface membrane blebs allowed us to measure the lateral diffusion of lipids in the membrane without the influence of underlying cytoskeletal structures. The restricted diffusion found on the normal surface membrane of mature parasites was found to be released in membrane blebs. Quenching of fluorescent lipids on blebs indicated that all probes were present almost entirely in the external monolayer. Juvenile worms exhibited lower lateral diffusion coefficients than mature parasites: in addition, the lipids partitioned into the external monolayer. The results are discussed in terms of membrane organization, cytoskeletal contacts, and biological significance.

Animals↗
PubMed1986

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.

ATP-Binding Cassette Transporters↗
PubMed1985

The influence of lipid composition and lectin-glycophorin interaction on the rotational diffusion of glycophorin in vesicles, as measured by time-resolved phosphorescence depolarization.

The rotational mobility of glycophorin in various lipid vesicles was studied, using time-resolved measurements of the depolarization of laser flash excited phosphorescence of glycophorin labelled with the triplet probe erythrosin. With the exception of dimyristoylphosphatidylcholine at the phase transition no phosphorescence depolarization decays were observed in the 1-300 microseconds time interval following the laser flash. Instead, a constant anisotropy level was observed, with two distinct values depending on the experimental system. In liquid-crystalline bilayers of dioleoylphosphatidylcholine, bovine brain phosphatidylserine and dimyristolyphosphatidylcholine, the anisotropy was 0.01. This was increased to 0.03 upon addition of wheat germ agglutinin which aggregates glycophorin. In the case of gel state dimyristoylphosphatidylcholine and liquid-crystalline dioleoylphosphatidylethanolamine the anisotropy also amounted to 0.03. Experiments with glycerol to vary the viscosity of the medium, and theoretical considerations, exclude the possibility that these different anisotropy levels are related to differences in motional properties of the entire protein/lipid vesicles. These results strongly suggest that the anisotropy level of 0.03 corresponds to slowly rotating glycophorin (rotational relaxation time greater than 0.3 ms) while the anisotropy level of 0.01 corresponds to fast rotating glycophorin (rotational correlation time less than 1 microseconds). The difference in glycophorin mobility is discussed in terms of aggregation state of the protein, lipid composition of the vesicle bilayer and membrane viscosity. The observed differences in rotational mobility of glycophorin in glycophorin/dioleoylphosphatidylcholine vesicles, glycophorin/bovine heart phosphatidylserine vesicles as compared to glycophorin/dioleoylphosphatidylethanolamine vesicles are not in quantitative agreement with the relative size of the intramembrane particles in these systems as revealed by freeze-fracture electron microscopy.

Animals↗
PubMed1984

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion.

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.

Carcinoma, Squamous Cell↗
PubMed1983

Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy.

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations [( Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.

Aminoquinolines↗
PubMed1983

Segmental motion and rotational diffusion of the Ca2+-translocating adenosine triphosphatase of sarcoplasmic reticulum, measured by time-resolved phosphorescence depolarization.

We studied the rotational mobility of the Ca2+ + Mg2+-activated ATPase in skeletal-muscle sarcoplasmic-reticulum vesicles, using time-resolved measurements of the depolarization of laser-flash-excited phosphorescence of the extrinsic triplet probe erythrosin. Our results are in general agreement with those of others [Bürkli & Cherry (1981) Biochemistry 20, 138-145] obtained by linear dichroism methods. In addition, we directly observed fast depolarization in the 1-5 microseconds time range that can be attributed to limited motion of part of the protein (segmental motion). Temperature-dependent changes in phosphorescence anisotropy indicated the onset of a conformational change in structure of the Ca2+ + Mg2+-activated ATPase at 11-13 degrees C. We also describe the synthesis of 5-iodoacetamidoerythrosin.

Adenosine Triphosphatases↗
PubMed1983

Carbocyanine dyes used as fluorescent triplet probes for measuring slow rotational diffusion of lipids in membranes.

A method for measuring the slow rotational diffusion of lipids or lipid domains in membranes has been developed. It covers the time range from 20 microseconds-5 ms, and has a greater than 5 x 10(4) molecules of probe. The method uses acyl-substituted carbocyanine dyes as fluorescent triplet probes and a laser-microscope combination for excitation and measurement of the triplet state. Rotation rates in dimyristoylphosphatidylcholine vesicles were sensitive to the liquid-to-gel transition. Slow rotations with relaxation times of about 100 microseconds were detected at the transition temperature region.

Carbocyanines↗